The transthyretin (TTR) amyloidoses, including senile systemic amyloidosis, familial amyloid cariomyopathy, and familial amyloid polyneuropathy, are characterized by extracellular amyloid deposits composed of TTR. This protein normally functions as a homotetramer to transport thyroid hormones and holo- retinol binding protein in the blood and cerebrospinal fluid. The details of TTR aggregation in vitro are well understood, but these assays require non-physiological conditions, such as low pH, to induce tetramer dissociation, monomer destabilization, and subsequent aggregation on a laboratory time scale. The causes for TTR aggregation under physiological conditions remain poorly understood. Approach: Amyloid fibrils extracted from patient tissue contain both protein and lipid components, and lipids have been shown to influence the aggregation in vitro of many amyloidogenic proteins including TTR. However, the mechanisms underlying these effects have yet to be determined. Our preliminary results show dramatic differences between the effects of various lipids on the aggregation of TTR. We will examine the structural and mechanistic alterations that underlie TTR aggregation in the presence of lipids, and determine the extent to which these alterations influence cytotoxicity. Hypothesis: The central hypothesis of this proposal is that certain membrane lipids alter the kinetics and pathway of TTR aggregation through distinct mechanisms, resulting in discrete aggregate structures that differentially influence cellular toxicity. Study Design: In Specific Aim 1, we will investigate the mechanisms underlying the observed lipid-specific effects on TTR aggregation. Structural and biophysical assays will be used to investigate alterations in the secondary, tertiary, and/or quaternary structure of TTR when aggregated in the presence of lipids. Disease- associated TTR variants with distinct kinetic and thermodynamic properties will be used to assess the specific steps in the aggregation process at which lipids exert their influence. In addition, we will measure the extent of lipid incorporation into the TTR aggregates. In Specific Aim 2, we will examine the relative cytotoxicity of TTR aggregates formed in the presence of various lipids. Cultured primary cells and/or immortalized cell lines from several tissues will be used to detect cell-type-specific effects. Global cytotoxicity will be measured, as well as specific markers for various types of cell stress. Taken together, the experiments proposed here will shed light on the mechanism(s) by which lipids accelerate TTR aggregation.